Metagenomic analysis of Neandertal bone Vi33.16 sequences.

BACKGROUND: The Neandertal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to < 6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized. PRINCIPAL FINDINGS: Our taxonomic profiling based on the analyses of 3.95 Gb of Neandertal DNA isolated from the Vindija Neandertal Vi33.16 fossil showed that 90% of about 50,000 rRNA sequence reads were of bacterial origin and that actinobacteria accounted more than 75% of the bacterial rRNA sequences. Likewise, actinobacteria represented more than 80% of the PCR-amplified 16S rRNA sequences from a cave sediment sample taken from the same G layer as the Neandertal bone. We assembled all 16S rRNA sequence read assigned to Streptomycetales, and analysed the patterns of nucleotide differences in the individual sequence reads compared to assembled consensus sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neandertal rRNA sequences, but not for the Streptomyces-like rRNA sequences. SIGNIFICANCE: Our analyses suggest that actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neandertal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from cave sediment bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial communities and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures, such as selective degradation of bacterial DNA with particular base composition patterns, to increase the relative amount of ancient DNA in the sample.